title: Rapamycin inhibits the production of myofibroblasts and reduces corneal scarring after photorefractive keratectomy.
abstract: OBJECTIVE
  Corneal stromal scarring partly involves the production of corneal myofibroblasts. The purpose of this study was to examine the effects of rapamycin (an inhibitor of the mammalian target of rapamycin [mTOR] pathway) on myofibroblast formation in vitro and in-vivo.
  
  
  METHODS
  Human corneal fibroblasts were grown in culture and transformed into myofibroblasts using TGF-β (2 ng/mL). The phosphorylation (activation) of the mTOR pathway was examined by immunoblotting. Cell proliferation with and without rapamycin was examined by thiazolyl blue tetrazolium bromide (MTT) assay and Ki67 staining. The expression of the myofibroblast differentiation marker smooth muscle actin (SMA) was examined by immunostaining and immunoblotting. The functional effects of rapamycin were measured using a gel contraction assay. For in vivo studies, 140 μm laser ablation was performed on rabbit corneas followed by subconjunctival rapamycin or vehicle. Corneal haze development was graded at 4 weeks, while the expression of myofibroblast markers was examined by immunostaining and immunoblotting.
  
  
  RESULTS
  The TGF-β activated the mTOR pathway with peak phosphorylation at 2 to 4 hours. Treatment of corneal fibroblasts with rapamycin reduced their proliferation by 46% compared to control. Rapamycin significantly inhibited TGF-β-induced expression of myofibroblast markers (17.2% SMA positive cells with rapamycin compared to 69.0% in control). Rapamycin also significantly inhibited TGF-β-induced collagen gel contraction. In the rabbit eyes treated with rapamycin, corneal haze development was significantly less compared to controls (0.75 ± 0.4 vs. 2.17 ± 0.7).
  
  
  CONCLUSIONS
  Rapamycin appears to inhibit proliferation and differentiation of corneal myofibroblasts and, thus, may provide an effective therapeutic measure for preventing corneal scarring.
authors.affiliation: Illinois Eye and Ear Infirmary, University of Illinois at Chicago (UIC) Department of Ophthalmology and Visual Sciences, Chicago, Illinois.
authors.forename: Behrad Y
authors.initials: BY
authors.lastname: Milani
authors.forename: Farnoud Y
authors.initials: FY
authors.lastname: Milani
authors.forename: Dong-Wouk
authors.initials: DW
authors.lastname: Park
authors.forename: Abed
authors.initials: A
authors.lastname: Namavari
authors.forename: Jarna
authors.initials: J
authors.lastname: Shah
authors.forename: Hossein
authors.initials: H
authors.lastname: Amirjamshidi
authors.forename: Hongyu
authors.initials: H
authors.lastname: Ying
authors.forename: Ali R
authors.initials: AR
authors.lastname: Djalilian
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002454:Cell Differentiation / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D002921:Cicatrix / Q000378:metabolism / Q000473:pathology / Q000517:prevention & control*; D003318:Corneal Opacity / Q000473:pathology / Q000601:surgery*; D003319:Corneal Stroma / Q000378:metabolism / Q000473:pathology* / Q000601:surgery; D004195:Disease Models, Animal; D005260:Female; D006801:Humans; D007166:Immunosuppressive Agents / Q000494:pharmacology; D058628:Myofibroblasts / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D018903:Photorefractive Keratectomy; D011817:Rabbits; D020123:Sirolimus / Q000494:pharmacology*; D016212:Transforming Growth Factor beta / Q000378:metabolism
chemical_list: D007166:Immunosuppressive Agents; D016212:Transforming Growth Factor beta; D020123:Sirolimus
keywords: corneal haze; excimer laser; rapamycin